6 (R)-l-erythro-5,6,7,8-tetrahydrobiopterin receptor

ABSTRACT

A receptor (binding protein) specific for 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin which is a drug effective for neuropsychiatric disorders such as infantile autism is provided. This receptor is contained in the cytosol fraction or the membrane fraction obtained from a homogenate of an animal organ. It is solubilized from the membrane fraction in the presence of 3-{(3-cholamidopropyl)-dimethylammonio}-1-propanesulfonate (CHAPS). This receptor is characterized in that the binding ability thereof to 6R-BH4 is mostly lost by heating at 100° C. for 10 minutes, the binding ability thereof to 6R-BH4 is significantly lost by incubating with trypsin at 37° C. for 15 minutes, and said receptor shows a relative molecular weight of about 340 to 380 kilodaltons when specifically bound to tritium-labeled 6R-BH4 and analyzed with a molecular sieve provided with a COSMOSIL 5 Diol-300 column.

FIELD OF THE INVENTION

This invention relates to a receptor which is specific for6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin which will hereinaftersometimes be referred to as 6R-BH4 while the one labeled with tritium atthe 6-position will sometimes be referred to as [6-³ H]6R-BH4. Morespecifically, the invention relates to a 6R-BH4 receptor which isuseful; as a tool in studying biochemical cellular reactions in responseto 6R-BH4 binding to the cells; as a tool in searching for anddeveloping agonists of the 6R-BH4 actions on the central nervous andimmunological systems or antagonists capable of interrupting theseactions when said search and development are conducted on the basis ofthe biochemical cellular reactions in response to 6R-BH4 binding to thecells and various other natures concerning the binding ability of6R-BH4; and also as a diagnostic or therapeutic drug for diseases whichare attributable to 6R-BH4.

BACKGROUND OF THE INVENTION

6R-BH4 is known to be a common coenzyme for tyrosine hydroxylase andtryptophan hydroxylase which are rate-limiting enzymes in thebiosynthesis of neurotransmitters such as dopamine and serotonin. It isconsidered that 6R-BH4 is a regulating factor in the biosynthesis ofthese neurotransmitter amines since 6R-BH4 is contained in nerve endingsonly in an amount which is approximately the Km value of eachhydroxylase. In fact, a shortage or decrease in enzymes whichparticipate in the biosynthesis of this coenzyme from GTP will give riseto a decrease in neurotransmitter amines, thus resulting in variousneuropsychiacric diseases. Actually, malignant phenylketonuria wasdiscovered in 1974 and subsequent studies have revealed that autopsiedbrain specimens and cerebrospinal fluid of patients with Parkinson'sdisease and Alzheimer's dementia show a decrease In 6R-BH4 content. Ithas also been found that supplementation therapy by 6R-BH4 is effectivein the treatment of malignant phenylketonurla, juvenile Parkinson'sdisease and, in accordance with recent studies, infantile autism of thedysbolism type. Based on these facts, over a hundred 6R-BH4 derivativeshave been drug-designed in Switzerland, the United States and Japan andscreened for their coenzyme action analogous to 6R-BH4. However, therehas not been discovered any derivative which is superior to 6R-BH4 interms of its action. In addition, no finding has been made hithertorelating to the biochemical cellular responses of 6R-BH4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an HPLC chromatogram of standard samples provided foridentification of the labeled compound shown in FIG. 2;

FIG. 2 is an HPLC chromatogram carried out for confirming theradiochemical purity of the purified synthetic [6-³ H]6R-BH4, i.e., aspecimen finally employed in the binding test;

FIGS. 3a and 3b are graphs showing the binding abilities of fractionsprepared from various regions of rat brain and other tissues, wherein(a) shows the results of P2 fraction and (b) shows the results ofcytosol fraction;

FIG. 4 is a graph showing the radioactivities which bound to thereceptor when [6-³ H]6R-BH4 was added at various concentrations to adetermined amount of the solubilized receptor;

FIG. 5 is a graph drawn by Scatchard-plot analysis of the data of thespecific binding shown in FIG. 4;

FIG. 6 is a graph showing that the receptor of the present invention hasa specific binding ability to 6R-BH4; and

FIG. 7 is a gel filtration chromatogram of the solubilized fractioncontaining the receptor of the present invention.

DISCLOSURE OF THE INVENTION

Our recent studies have resulted in a novel finding which demands agreat change in the concepts relying on previous knowledge.

That is to say, it has been discovered that 6R-BH4 enhances the releaseand liberation of neurotransmitter amines such as dopamine (DA),norepinephrine and serotonin, and that it also enhances the liberationof glutamir acid, aspartic acid or γ-aminobutyric acid (GABA) via DA andthe release of acetylcholine via serotonin. On the other hand, it hasbeen also discovered that when the endogenous 6R-BH4 level is lowered byinhibiting the biosynthesis of 6R-BH4, the DA level per se is notsignificantly changed whereas the release of DA is suppressed to 50 orlower.

These findings suggest that the endogenous 6R-BH4 may trigger somechange in the mechanism of DA release. Thus we have conducted extensiveinvestigations in order to clarify the molecular mechanism of DArelease. As a result, we have discovered, in a membrane fraction of thebrain, a receptor site which is saturated by 6R-BH4 and highly specificfor 6R-BH4 with high affinity and hence is the possible regulatory sitein the DA release mechanism. We have further found that because of saidspecificity, 6R-BH4 receptor is capable of strictly distinguishing 6R-and 6S-optical isomers of tetrahydrobiopterin and thus is capable ofselectively recognizing the 6R-isomer. Thus, these characteristics ofthe receptor completely differ from those of certain known enzymescapable of recognizing 6R-BH4 as their coenzyme.

Accordingly, the present invention relates to a receptor specific for6R-BH4.

The receptor of the present invention specifically reacts with 6R-BH4.Further, it is solubilized with CHAPS[3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate]. When it isheated at 100° C. for 10 minutes, the solubilized product loses most ofits binding ability to 6R-BH4. When incubated with trypsin at 37° C. for15 minutes, the solubilized product significantly loses its bindingability to 6R-BH4. The receptor has a relative molecular weight about340 to 380 kilodaltons when a rat cerebellum specimen solubilized andlabeled with [6-³ H]6R-BH4 is separated by a molecular sieve column onCOSMOSIL 5 Diol-300 porous silica, 300 Å, and the radioactivity ismeasured. As a result of an analysis by SDS electrophoresis, it has amolecular weight of about 160 kilodaltons. The receptor of the presentinvention may exist as a molecular fragment specifically reactive to6R-BH4.

The above-mentioned properties of the 6R-BH4 receptor according to thepresent invention are confirmed by the following methods.

Binding to 6R-BH4

The action as the receptor for 6R-BH4 can be confirmed by studying theability to bind to 6R-BH4.

For example, fractions obtained by homogenizing various regions sectionsof rat brain or other organs can be suspended in an appropriate buffersolution, for example, 50 mM Tris hydrochloride (pH 7.4), 100 mM NaCl,10 mM MgCl₂, 2 mM ascorbic acid and 6 mM cysteine, and incubatedtogether with an appropriate amount of tritium-labeled6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin ([6-³ H]6R-BH4) underappropriate conditions, for example, at 37° C. for 10 minutes. Next, theprotein-bound fraction is separated by a glass filter and theradioactivity in this fraction is determined by a conventional method.

High affinity to 6R-BH4

The high affinity of the receptor to [6-³ H]6R-BH4 can be confirmed byScatchard plot analysis.

The Scatchard plot analysis may be carried out, for example, as follows.In an appropriate buffer solution, for example, 50 mM Tris hydrochloride(pH 7.4), 100 mM NaCl, 10 mM MgCl₂, 2 mM ascorbic acid and 6 mM cysteinesolution, a fraction containing the receptor solubilized with CHAPS isincubated with [6-³ H]6R-BH4 at various concentrations under appropriateconditions, for example, at 37° C. for 10 minutes. Then theradioactivity is measured by a conventional method and plotted. From theplots, Kd (dissociation constant) and Bmax are determined and employedas indicators of the affinity. The receptor according to the presentinvention shows an affinity exceeding those of known enzymes taking partin the 6R-BH4 biosynthesis or enzymes for which 6R-BH4 serves as acoenzyme.

Binding specificity to 6R-BH4

The specificity of the receptor of the present invention in binding to6R-BH4 can be confirmed by a competitive binding test with the use ofvarious compounds at varying concentrations.

In this method, the above-mentioned compounds at varying concentrations(for example, biopterin as will be described in Example 6 hereinafter)are added as a ligand simultaneously with [6-³ H]6R-BH4 to the reactionmixture and the reaction is performed. The ability of the receptor ofthe present invention to bind to 6R-BH4 will not be inhibited to anysubstantial extent by these ligands.

Characteristics as Protein

1) Inactivation Under Heating

A solution of a receptor fraction solubilized with CHAPS in 10 mM CHAPSand 50 mM Tris hydrochloride (pH 7.4) is subjected to treatment underheating at 100° C. for 10 minutes. Then the same binding test as the onedescribed above is carried out to confirm disappearance of the bindingability.

2) Inactivation by Protease

To a solution of a receptor fraction solubilized with CHAPS in 10 mMCHAPS and 50 mM Tris hydrochloride (pH 7.4), 100 μg/ml of trypsin (WakoPure Chemical Industries, Ltd.) is added and the mixture is incubated at37° C. for 15 minutes. Next, the same binding test as the one describedabove is carried out in order to confirm disappearance of the bindingability.

3) Gel Filtration HPLC Analysis of Receptor Protein Labeled With [6-³H]6R-BH4

In a solution of 100 mM calcium phosphate buffer, 100 mM NaCl, 10 mMMgCl₂, 2 mM ascorbic acid and 6 mM cysteine, a fraction of the receptorsolubilized with CHAPS is incubated together with an appropriate amountof [6-³ H]6R-BH4, in a reaction volume of 120 μl, at 37° C. for 10minutes. The reaction mixture thus obtained is injected into HPLC underconditions as specified below.

Column: Cosmoseal 5 Diol-300 (7.5×300 mm),

Mobile phase: 100 mM potassium phosphate buffer solution, pH 7.4, 5 mMCHAPS, 0.1 mM ascorbic acid and 0.1 mM cysteine.

Flow rate: 1 ml/min.

The eluate is fractionated at the rate of 0.25 ml/15 sec/test tube.After adding 16 ml of creasol to each fraction, the radioactivity ismeasured in a liquid scintillation counter. Then the receptor peak iscompared with molecular weight markers to confirm the relative molecularweight. In this HPLC analysis, the receptor of the present inventiontypically shows a single peak and a relative molecular weight of about340 to 380 kilodaltons.

4) Analysis by SDS Electrophoresis

A receptor protein fraction from rat cerebellum, which has beensolubilized by CHAPS and partially purified by molecular sieve columnchromatography followed by 2',5'-ADP affinity column chromatography, iselectrophoresed on an SDS polyacrylamide gel in a conventional mannerand the separated bands are observed under silver-staining. Main bandsare located around 50 to 60 kilodaltons and at around 160 kilodaltons.The bands at 50 to 60 kilodaltons are assignable to glutathionereductase and malic enzyme which have affinity to the 2',5'-ADP columnbut never bind to 6R-BH4. Thus the molecular species at 160 kilodaltonscorresponds to the protein (receptor) which binds to 6R-BH4.

The 6R-BH4 receptor of the present invention can be extracted andpurified from animals and plants, for example, tissues of mammalsincluding humans or insects. Although the cerebellum which contains alarge amount of the receptor of the present invention is particularlysuitable isolation source thereof, there are a number of tissuescontaining this receptor, as will be shown in Example 4 hereinafter.Furthermore, the receptor of the present invention may occur as afragment specifically reactive with 6R-BH4 in the molecules of certainbioproteins. It may, therefore, be possible to obtain the receptor fromsuch proteins.

The 6R-BH4 receptor of the present invention can be prepared from anisolation source by any appropriate method without particularlimitation. For example, an isolation source such as various regions ofrat brain or other organs is homogenized and spun at about 1,000×g. Thesupernatant is centrifuged at about 17,000×g. The receptor of thepresent invention is contained in the precipitate (membrane fraction)and in the supernatant (cytosol fraction). The centrifugation may berepeated twice or more, if necessary. The receptor contained in theprecipitate can be solubilized in the presence of an appropriatesurfactant such as CHAPS, which can be separated by an appropriate meanssuch as centrifugation to provide a solubilized product. It is possibleto further purify the solubilized product, with the binding ability to6R-BH4 as an indicator, by an appropriate combination of purificationprocedures such as a gel filtration, an ion exchange chromatography, theuse of various adsorbents including immunoadsorbents andelectrophoresis. A specific purification example will be given inExample 7 hereinafter.

The fraction which is specifically reactive with 6R-BH4 can be subjectedto a digestion with a conventional protease such as lysyl endopeptidaseor to treatment with a chemical substance such as cyanogen bromidewhereby the receptor peptide fragment can be obtained. The peptidefragment may be further purified by, for example, HPLC. The fragment maybe screened by a competitive binding test for 6R-BH4.

Alternatively, the receptor of the present invention can be obtained bythe method comprising the steps of extracting messenger RNA (mRNA) fromcells of an organ abounding in said receptor such as the cerebellum, thewhole brain, heart, spleen, lung or adrenal glands by a conventionalmethod; constructing a cDNA library from the mRNA; selecting a cDNAencoding the amino acid sequence of the target protein by screening thelibrary by any of various procedures; inserting the cDNA thus selectedinto an expression vector in a conventional manner; transfecting saidvector into appropriate host cells (animal cells); and then allowingexpression of the protein. The above-mentioned selection of the cDNA canbe conducted by, for example, inserting the cDNAs into a vector capableof functioning in animal cells, transfecting host animal cells with saidvector and then detecting the target cDNA based on the specific bindingbetween the receptor expressed on the surface of the host cells and6R-BH4.

When the receptor of the present invention is to be obtained by geneticengineering techniques, not only a recombinant gene originating from thecDNA but also a synthetic DNA obtained by designing the nucleotidesequence based on the amino acid sequence which will be deduced by thecDNA may be used.

The receptor of the present invention is useful as a tool in studyingbiochemical cellular reactions in response to 6R-BH4 binding to thecells; as a tool in searching for and developing agonists of the 6R-BH4actions on the central nervous and immunological systems or antagonistscapable of interrupting these actions when said search and developmentare conducted on the basis of the biochemical cellular reactions inresponse to 6R-BH4 binding to the cells and various other naturesconcerning the binding ability of 6R-BH4; and also as a diagnostic ortherapeutic drug for diseases which are attributable to 6R-BH4.

Particular examples of the diagnostic and therapeutic drugs includediagnostic drugs for diseases induced by shortage, decrease or increasein 6R-BH4 such as malignant phenylketonuria, Parkinson's disease,infantile autism, manic-depressive psychosis and Alzheimer's disease andtherapeutic drugs for diseases in which the receptor can effectivelyinduce pharmacological effects by the inhibitory action on thebiochemical cellular reactions responsive to binding of 6R-BH4 to thereceptor.

For these purposes, the receptor of the present invention can be used invarious assay methods in a conventional manner taking advantage of thespecific binding ability thereof to 6R-BH4. As examples of such assaymethods are mentioned, assays on the basis of competition, for bindingto a labeled 6R-BH4 standard, between the receptor of the invention andand samples (for example, biological samples such as plasma, autopsiedbrain and cerebrospinal fluid or candidates of an agonist or anantagonist), and specific binding tests between the receptor of thepresent invention and a labeled sample. In these assay procedures, thereceptor of the present invention may be immobilized on a solid carrierin accordance with a conventional method.

The receptor of the present invention can be provided in the form of akit to be used in the above-mentioned tests. The receptor may beprovided in the form of either a solution, a freeze-dried product or animmobilized product on a solid carrier. Further, the kit may optionallycontain a labeled 6R-BH4 standard and other components required in theassay such as a buffer commonly used for diluting samples, a buffer fordiluting various reagents and a washing liquid.

The following examples are provided to further illustrate the presentinvention. However, it is to be understood that the present invention isnot limited thereto.

EXAMPLE 1 Production of Tritium-Labeled 6R-BH4

In accordance with the following reaction scheme, tritium-labeled 6R-BH4(hereinafter referred to as [6-³ H]6R-BH4) was prepared.

Tritium was introduced to the 6-position of 7,8-dihydrobiopterin (I) viaa reduction reaction with NaB[³ H]₄. Then proton was introduced into the5-position by a water-quenching reaction to give [6-³ H]6R-BH4 (II).##STR1##

The obtained racemic mixture was purified by HPLC and the target [6-³H]6R-BH4 was separated from [6-³ H]6S-BH4. FIG. 1 shows an UVchromatogram (265 nm) of the standard sample. The results shown in FIG.2 indicate that the radiochemical purity of the [6-³ H]6R-BH4 finalproduct was 98% or higher.

EXAMPLE 2 Preparation of P2 Fraction and Cytosol Fraction

By using various regions of rat brain and other organs as startingmaterials, fractions containing 6R-BH4 receptor were prepared in thefollowing manner.

The starting material was homogenized in 10 volumes of 0.32M sucrosesolution and then centrifuged (1,000×g, 12 minutes). The supernatant wasseparated and further centrifuged at a high speed (17,000×g, 30minutes). The precipitate was suspended in 10 volumes of 50 mM Trishydrochloride (pH 7.4). The obtained suspension was further centrifuged(17,000×g, 10 minutes) and the precipitate thus obtained was suspendedagain in 10 volumes of 50 mM Tris hydrochloride (pH 7.4). The suspensionthus obtained was referred to as P2 fraction.

The starting material was homogenized in 10 volumes of 0.32M sucrosesolution and then centrifuged (1,000×g, 12 minutes). The supernatant wasseparated and further centrifuged at a high speed (17,000×g, 30minutes). The supernatant was further centrifuged (100,000×g, 60minutes) and the supernatant thus obtained was referred to as thecytosol fraction.

EXAMPLE 3 Preparation of Solubilized Fraction

By using various regions of rat brain and other organs as a startingmaterial, a solubilized fraction containing 6R-BH4 receptor was preparedin the following manner.

The starting material was homogenized in 10 volumes of 0.32M sucrosesolution and then centrifuged (1,000×g, 12 minutes). The supernatant wasseparated and further centrifuged at a high speed (17,000×g, 30minutes). The precipitate was suspended in 10 volumes of 50 mM Trishydrochloride (pH 7.4). The suspension was further centrifuged(17,000×g, 10 minutes) and the precipitate thus obtained was suspendedin 5 volumes of 5 mM CHAPS and 50 mM Tris hydrochloride (pH 7.4). Thesuspension was stirred in a refrigerated room for 1 hour and thenultracentrifuged (100,000×g, 60 minutes). The supernatant thus obtainedwas referred to as the solubilized fraction.

EXAMPLE 4 Binding of 6R-BH4 to P2 and Cytosol Fractions

The ability of [6-³ H]6R-BH4 to bind to P2 and cytosol fractionsprepared in Example 2 was determined. As a result, it was confirmed that6R-BH4 receptor was contained in P2 fraction and cytosol fractionderived from all of the tested brain regions and organs.

METHOD

In a suspension of 50 mM Tris hydrochloride (pH 7.4), 100 mM NaCl, 10 mMMgCl₂, 2 mM ascorbic acid and 6 mM cysteine, P2 fraction was incubatedwith 20 nM [6-³ H]6R-BH4 at 37° C. for 10 minutes in a reaction volumeof 200 μl or 400 μl. Then the reaction was ceased by adding 4 ml ofice-cooled 50 mM Tris hydrochloride (pH 7.4) and 100 mM NaCl.

Then the mixture was passed through a glass filter. After being washed 4times with 4 ml portions of 50 mM Tris hydrochloride (pH 7.4) and 100 mMNaCl, the filter was taken off and filled with 2 ml of ethanol and 8 mlof creasol (Nacalai Tesque). Then the radioactivity on the filter wasmeasured in a liquid scintillation counter.

FIG. 3 shows the results in terms of "mean±standard deviation (S.D.)"(n=4). The specific binding represents the binding wherein thenonspecific binding as determined by reacting 10 μM of 6R-BH4 and [6-³H]6R-BH4 has been subtracted.

FIG. 3 clearly indicates that the fractions derived from cerebellumshowed higher levels of binding. Thus, the following description will bemade by referring to P2 fraction and CHAPS-solubilized fractions fromcerebellum.

In the case of cytosol fraction, the fraction was incubated in thesolution of the same composition as the above-mentioned one employed forP2 fraction at 37° C. for 10 minutes. Then 3 mg/ml (final concentration)of bovine γ-globulin and 15% (final concentration) of polyethyleneglycol 6000 were added to cease the reaction.

Next, the reaction mixture was cooled on ice for 10 minutes and thenpassed through a glass filter. After being washed 4 times with 4 mlportions of 8% polyethylene glycol and 10 mM Tris hydrochloride (pH7.4), the filter was taken off and placed in a vial. 2 ml of ethanol and8 ml of creasol (Nacalai Tesque) were added to the filter and theradioactivity on the filter was measured in a liquid scintillationcounter.

The distribution of 6R-BH4 receptor in the brain cytosol fraction wassimilar to that of the P2 fraction.

EXAMPLE 5 Scatchard Plot Analysis

By using a determined amount of the solubilized fraction of ratcerebellum, Scatchard plot analysis with regard to the solubilizedreceptor protein of the present invention was performed by theconventional method. The following data was obtained: Kd (dissociationconstant)=21 nM, Bmax (maximum binding constant)=550 fmol/mg protein.The Kd value thus obtained was greater than those (several ten μM) ofany known enzymes which participate in the biosynthesis of 6R-BH4 orenzymes for which 6R-BH4 acts as a coenzyme. Thus it was confirmed thatthe receptor according to the present invention was a completely novelone.

METHOD

About 0.6 mg (protein) of the solubilized fraction obtained in Example 3was incubated together with [6-³ H]6R-BH4 at various concentrations in a400 μl solution of 50 mM Tris hydrochloride (pH 7.4), 100 mM NaCl, 10 mMMgCl₂, 2 mM ascorbic acid and 6 mM cysteine at 37° C. for 10 minutes.Then, 3 mg/ml (final concentration) of bovine γ-globulin and 15% (finalconcentration) of polyethylene glycol 6000 were added to cease thereaction.

The reaction mixture was cooled on ice for 10 minutes and then filteredthrough a glass filter. After being washed 4 times with 4 ml portions of8% polyethylene glycol and 10 mM Tris hydrochloride (pH 7.4), the filterwas taken off and placed in a vial. 2 ml of ethanol and 8 ml of creasol(Nacalai Tesque) were added to the filter and the radioactivity on thefilter was measured in a liquid scintillation counter.

The results are shown in FIGS. 4 and 5. FIG. 4 is a graph showing theradioactivities which bound to the receptor when [6-³ H]6R-BH4 was addedat various concentrations to a determined amount of the solubilizedreceptor. In FIG. 4, these radioactivities are referred to as the totalbinding, while nonspecific binding indicates radioactivities measured byadding 10 μM of unlabeled 6R-BH4 simultaneously with [6-³ H]6R-BH4 atvarious concentrations. Accordingly, the saturation of the specificbinding can be calculated as the difference between the total andnonspecific bindings. FIG. 5 is a graph drawn by Scatchard plot analysisof the data of the specific binding in FIG. 4.

EXAMPLE 6 Binding Specificity

By using the solubilized fraction of rat cerebellum, a competitivebinding test between various compounds at varying concentrations and 13nM of [6-³ H]6R-BH4 was carried out. As a result, it was found that thereceptor of the present invention is specific for 6R-BH4.

IC₅₀ data indicated that the affinity of the receptor of the presentinvention to 6R-BH4 was more than 50 times and 1,000 times as high asits affinities to 7,8-dihydrobiopterin (BH2) and 6S-BH4, respectively.This is a point of great difference from the previous knowledge that thecoenzyme activity of 6R-BH4 for aromatic amino acid hydroxylases ismaintained relatively well even if 6R-BH4 is converted to 6S-BH4.

These results also support the fact that the receptor of the presentinvention is specific for 6R-BH4. In addition, since this receptor doesnot show any substantial binding ability to tetrahydrofolic acid (FAH4),it completely differs From various known folic acid-binding proteinsoccurring in vivo.

METHOD

About 0.3 mg (protein) of the solubilized fraction obtained in Example 3was incubated together with 13 nM of [6-³ H]6R-BH4 in a 200 μl solutionof 50 mM Tris hydrochloride (pH 7.4), 100 mM NaCl, 10 mM MgCl₂, 2 mMascorbic acid and 6 mM cysteine at 37° C. for 10 minutes. Then 3 mg/ml(final concentration) of bovine γ-globulin and 15% (final concentration)of polyethylene glycol 6000 were added to cease the reaction.

Next, the reaction mixture was cooled on ice for 10 minutes and thenpassed through a glass filter. After being washed 4 times with 4 mlportions of 8% polyethylene glycol and 10 mM Tris hydrochloride (pH7.4), the filter was taken off and placed in a vial. 2 ml of ethanol and8 ml of creasol (Nacalai Tesque) were added to the filter and theradioactivity on the filter was measured in a liquid scintillationcounter.

The results thus obtained were referred to as total binding. Thenvarious compounds at varying concentrations were added as a ligandsimultaneously with [6-³ H]6R-BH4 in the above procedure, and thereaction was effected. FIG. 6 shows the results, wherein BP representsbiopterin, 6MePH4 represents 6-methyl-5,6,7,8-tetrahydropterin and DAHPrepresents 2,4-diamino-6-hydroxypyrimidine (a potential inhibitor forGTP cyclohydrolase, i.e., 6R-BH4 synthetase).

EXAMPLE 7 Characteristics as a Protein

1) Inactivation Under Heating

A solution of the solubilized fraction prepared in Example 3 in 10 mMCHAPS and 50 mM Tris hydrochloride (pH 7.4) was heated at 100° C. for 10minutes. Then the same binding test as the one described above wascarried out and the binding abilities were compared. As a result, mostpart (98%) of the binding ability of the solubilized protein to [6-³H]6R-BH4 disappeared.

2) Inactivation by Protease

To a solution of the solubilized fraction in 10 mM CHAPS and 50 mM Trishydrochloride (pH 7.4), 100 μg/ml of trypsin (Wako Pure ChemicalIndustries, Ltd.) was added and the mixture was incubated at 37° C. for15 minutes. Then the same binding test as the one described above wascarried out and the binding abilities were compared. As a result, thebinding ability of the solubilized protein to [6-³ H]6R-BH4significantly disappeared.

3) Gel Filtration HPLC Analysis of Receptor Protein Labeled with [6-³H]6R-BH4

As the result of an analysis with the use of a molecular sieve onCosmoseal 5 Diol-300 (Nacalai Tesque) column (refer to FIG. 7), [6-³H]6R-BH4 which had been bound to the receptor protein was detected at aposition around 340 to 380 kilodaltons between molecular markersthyroglobulin (670 kilodaltons) and γ-globulin (158 kilodaltons). Thelast peaks seen in FIG. 7 are considered to be assignable to unboundfree [6-³ H]6R-BH4 and ³ H₂ O formed during the treatment.

About 0.2 mg (protein) of the solubilized fraction obtained in Example 3was incubated together with about 360,000 dpm of [6-³ H]6R-BH4 in a 120μl solution of 100 mM potassium phosphate buffer (pH 7.4), 100 mM NaCl,10 mM MgCl₂, 2 mM ascorbic acid and 6 mM cysteine at 37° C. for 10minutes.

100 μl of the reaction product was injected into HPLC under theconditions as specified below.

Column: COSMOSIL 5 Diol-300 (7.5×300 mm).

Mobile phase: 100 mM potassium phosphate buffer solution, pH 7.4, 5 mMCHAPS, 0.1 mM ascorbic acid and 0.1 mM cysteine.

Flow rate: 1 ml/min.

The eluate was fractionated at the rate of 0.25 ml/15 sec/test tube.After adding 16 ml of creasol to each fraction, the radioactivity wasmeasured in a liquid scintillation counter. FIG. 7 shows the results.

Then the cytosol fraction was analyzed by the same method, and similarresults as the one described above were obtained.

4) Analysis by SDS Electrophoresis

Cerebellum fraction of a rat was solubilized with CHAPS and purifiedwith the above-mentioned COSMOSIL 5 Diol-300 column. Then the activefraction was further purified by using a 2',5'-ADP column (Pharmacia).From fractions showing high [6-³ H]6R-BH4 binding activities, about 3 μgof total protein was taken out and electrophoresed on an SDS-PAG plate4/20 (Daiichi Kagaku) and silver stained (Wako Pure Chemical Industries,Ltd.). As a result, main bands were located around 50 to 60 kilodaltonsand at about 160 kilodaltons. The bands at 50 to 60 kilodaltons wereassignable to glutathione reductase and malic enzyme which have affinityto 2',5'-ADP column but would never bind to 6R-BH4. Thus the molecularspecies at 160 kilodaltons corresponded to the 6R-BH4-binding protein(receptor).

We claim:
 1. An isolated and purified6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) receptor whichspecifically binds to 6R-BH4 and which is contained in the cytosolfraction obtained from a homogenate of an animal organ or in themembrane fraction obtained from a homogenate of an animal organ andwhich is capable of being solubilized from the membrane fraction in thepresence of 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate(CHAPS), characterized in that the binding ability thereof to bind to6R-BH4 is mostly lost by heating at 100° C. for 10 minutes, the bindingability thereof to bind to 6R-BH4 is significantly lost by incubatingwith trypsin at 37° C. for 15 minutes, and said receptor showssubstantially a single peak at about 340 to 380 kilodaltons when saidreceptor is solubilized from the membrane fraction in the presence ofCHAPS and is specifically bound to tritium-labeled 6R-BH4 and issubjected to gel filtration chromatography.
 2. The receptor of claim 1,wherein the binding of the receptor to6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin is between 50 to 1000 timesgreater than the binding of the receptor to 7,8-dihydrobiopterin or6(S)-L-erythro-5,6,7,8-tetrahydrobiopterin.
 3. An isolated and purifiedprotein which is contained in the cytosol fraction or in the membranefraction obtained from a homogenate of an animal organ and has aselective binding affinity to 6R-BH4, characterized in that the bindingability thereof to bind to 6R-BH4 is mostly lost by heating at 100° C.for 10 minutes, the binding ability thereof to bind to 6R-BH4 issignificantly lost by incubating with trypsin at 37° C. for 15 minutes,and said protein shows substantially a single peak at about 340 to 380kilodaltons when said protein is solubilized from the membrane fractionin the presence of CHAPS and specifically bound to tritium-labeled6R-BH4 and subjected to gel filtration chromatography.
 4. The protein ofclaim 3, wherein the binding of the protein to6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin is between 50 to 1000 timesgreater than the binding of the protein to 7,8-dihydrobiopterin or6(S)-L-erythro-5,6,7,8-tetrahydrobiopterin.